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Ds2645104515

  1. 1. International Journal of Modern Engineering Research (IJMER) www.ijmer.com Vol.2, Issue.6, Nov-Dec. 2012 pp-4510-4515 ISSN: 2249-6645Isolation and Molecular Characterization of Anti-Cancerous CompoundProducing Marine Bacteria by Using 16S rRNA Sequencing and GC-MS Techniques Sai Lakshman Mithun.V1, C.S.V.Ramachandra Rao2 Department of Biotechnology, DVR & Dr.HS MIC College of Technology, Kanchikacherla, Andhra Pradesh, India.Abstract: Extracts from microorganisms have served as a valuable source of diverse molecules in many drug discoveries.Identification of microbial strains having promising biological activities and purifying the bio-molecules which areresponsible for the biological activities, have led to the discovery of many bioactive molecules. Extracts of bacteria in vitrotested on various cancer cell lines. The lyophilized bacterial extract powder was dissolved in various chemical solvents likeMethanol, Chloroform and Ethyl acetate. From them cytotoxic assays was performed the extracts were screened on HCT 15and MES- SA cancer cell lines to study the cytotoxic potential. Where the Ethyl acetate showed the inhibition 87.34% whencompared to other solvents. The Ethyl acetate extract of isolate showed promising results by MTT assay and Trypan bluestaining. Identification of the microorganism, the selected isolates was characterized by using the 16s rRNA sequencingbased on microbial characterization, the compound produced by bacteria was analyzed by GC-MS technique. The organismwas identified as Micrococcus luteus and biochemical tests of the extracts were also carried out.Key words: Anticancer, Bacterial extracts, MTT assay, HCT 15 cell line, MES-SA cell line. I. INTRODUCTION Cancer is a disease in which a cell, or a group of cells represents uncontrolled growth invasion (intrusion on anddistortion of adjacent tissues), and metastasis (spread from one part to another part in the body through lymph or blood).These three malignant properties differentiate cancer from benign tumors, which are self-limited and do not invade ormetastasize while the malignant tumors are not self limited and metastasize. Most of cancers occur form a tumor; theoncology is deals with the study, diagnosis, treatment, and prevention of cancer and it’s a branch of medicine. Cancer is ahuman tragedy that affects people at all ages with the risk in most types increasing with age. Cancers are primarily anenvironmental disease with 90-95% of cases due to modification in lifestyle and environmental factors and 5-10% due togenetics Cancer is caused. By both external factors (tobacco, chemicals, radiation and infectious organisms) and internal factors (inheritedmutations, hormones, immune conditions and mutation that occurs from metabolism). Common environmental factorsleading to cancer death include: tobacco 25-30%, diet and obesity 30-35%, infections 15-20%, radiation, stress, lack ofphysical activity and environmental pollutants. [B Dhorajiya et al., 2011]. Early discovery of carcinogens by John Hill, an English physician, 1761. In it he made the first causal link betweensubstances in the environment and cancer when he described a relationship between tobacco snuff and nasal cancer. Thisbrought about the awareness of carcinogens (chemical agents that have been demonstrated to cause cancer). That associatedparticular occupations with an increased risk of developing specific forms of cancer the forerunner to the field of publichealth and cancer.[C. A. Almeida and S. A. Barry, 2010]. Natural products have played an important role in treating and preventing human diseases. Natural products withmedicinal value have come from various sources viz., terrestrial plants, terrestrial microorganisms, marine organisms, andterrestrial vertebrates and invertebrates the microbial extracts have served as a valuable source of diverse molecules in manydrug discovery efforts and led to the isolation of several important drugs. [Newman et al., 2002]. The chemical compositionof bioactive compounds of microbial origin is often highly complex. Organic compounds from aqua to terrestrialmicroorganisms have extensive use in the treatment of many diseases and serve as compounds of interest both in theirnatural form and as templates for synthetic modification. These compounds provided important contributions to thediscovery of antibacterial agents like penicillins, cephalosporins, tetracyclines, and polypeptides, immunosuppressive agentslike cyclosporine, ant diabetic agent like acarbose, cholesterol-lowering agents like lovastatin and mevastatin and anti canceragents like peplomycin, pentostatin, and epirubicin [Butler, 2005; Sneader, 2005] II. MATERIALS AND METHODSScreening and isolation of bacterial strains:Collection of soil sample: Marine soil samples were collected from Bay of Bengal coast of Machilipatnam, Krishna district, AP, India. About15 grams of soil sediments were collected in a sterile polythene bags and stored at 4 oC until further use. The soil sampleswere stored under sterile conditions for preventing the bacterial cross contamination. Then the sample was serially diluted totill 10-6 dilution by adding 1 gram of soil to 10ml of distilled water. www.ijmer.com 4510 | Page
  2. 2. International Journal of Modern Engineering Research (IJMER) www.ijmer.com Vol.2, Issue.6, Nov-Dec. 2012 pp-4510-4515 ISSN: 2249-6645Screening for bacterial single isolates: From the serially diluted samples, 100µl of sample was poured on Nutrient Agar plates (Himedia). From the 10 -5 -6and 10 dilutions the single isolated bacterial colonies were obtained on agar plates by the procedure according to [Shirai etal. (1989).], The bacterial pure cultures were isolated.Sub culturing of bacterial isolates: The inoculum was prepared by transferring a single colony of the isolates into 5 ml of the corresponding medium.This inoculum was transferred to 95 ml of the corresponding broth and bacterial isolates were incubated at c for 2 -3 days.this broth was used for the extraction of bioactive metabolites.Preparation of bacterial extracts: The culture broth was centrifuged at 3000 rpm for 15 min to obtain a clear supernatant. Components were extractedsuccessively dissolved with chemical solvents such as chloroform (C), Methanol (M) and Ethyl acetate (E) and followed byvacuum evaporation of these extracts to obtain the dry extracts by vacuum rotary evaporator. [Angel TreasaThomas etal.,2011]Sample preparation for biological studies: The bacterial crude extracts were dissolved in 1 ml DMSO and transferred to sterile vials of 2 ml capacity; thesesamples were stored at room temperature for further use, protected from light.Maintenance of cancer cell lines: Human colorectal adenocarcinoma Cancer cells (HCT 15) and Human uterine cancer cells (MES-SA) weremaintained in Dulbecco’s modified essential medium (DMEM) supplemented with 4.5 g/l glucose and 2mm 1-glutamine and5% fetal bovine serum (FBS) (growth medium) at 370c in 5% Co2 incubator.Trypan blue dye exclusion technique: A cell suspension was made with a fixed volume of cells (e.g. 1ml). Although an aseptic technique is not essentialin all stages of this procedure, 50uL of cell suspension was taken and mixed it with an equal volume of trypan blue. Solutionwas well mixed using a pipette. It transfers to a hemocytometer and then counted live cell as clear form and dead cell as bluecells. After staining with trypan blue solution counting should commence <5minutes as after that time the cells will begin totake up the dye. The hemocytometer was placed on the stage of an inverted microscope.Focus was adjusted and power until a single counting square fills the field. The number of cells per ml, and the total numberof cells were counted using the following formula:Calculate percent viability by using formula:% viability = (live cell count/total cell count) ×100Anticancer assay:Micro culture tetrazolium (MTT) assay: The monolayer cell culture was trypsinized and the cell count was adjusted to 1ml using medium. To each well of96 well microtitre plates, 0.1ml of diluted cell suspension was added. After 72 hour, the sample solution in wells was flickedoff and 50μl of MTT dye was added to each well. The plates were gently shaken and incubated for 4 hours at 37ₒ c in 5%CO2 incubator. The supernatant was removed, 50 μl of Propanol was added, and the plates were gently shaken to solubilizethe formed formazan. The absorbance was measured using a micro plate reader at a wavelength of 490 nm. The percentagegrowth inhibition was calculated using the Formula below:The percentage growth inhibition was calculated using following formula:%cell inhibition= 100-{(At-Ab)/ (Ac- Ab)} x100Where,At= Absorbance value of test compoundAb= Absorbance value of blankAc=Absorbance value of control [T. Mosmann, J. Immunol. Methods]Identification of microorganism: Biomass culture for anticancer activity assays was carried out in 500 mL conical flasks at 37°C temperature. TotalDNA was extracted and PCR amplification was performed according to the method described by [Fawley and Fawley(2004)] using following primers:Forward primer- 51 GGCGAACGGGTGAGTAA 31Reverse primer- 51 ACTGCTGCCTCCCGTAG 31 The Genomic DNA was isolated from the overnight grown bacterial culture was amplified with universal bacterialprimers. A 25μl of reaction mixture contains 15μl of master mix (10 x assay buffer, dntp’s, Taq polymerease and MgCl 2),1μl of forward primer, 1μl of reverse primer, 2μl of template DNA and 6μl of distilled water. PCR was carried out by thethermal cycler under the following conditions- An intialization step at 94ºC for 4min followed by 30 cycles of 94ºC for1min, 52ºC for 1min, 72ºC for 1.15min followed by final extension at 72ºC for 1min and holding temperature at 10ºC for www.ijmer.com 4511 | Page
  3. 3. International Journal of Modern Engineering Research (IJMER) www.ijmer.com Vol.2, Issue.6, Nov-Dec. 2012 pp-4510-4515 ISSN: 2249-66451min. The amplified DNA fragments were observed by agarose gel electrophoresis in 2% agarose gel and sequenced. Theunknown bacterium was identified usingGenBank database. The amplified PCR products were directly sequenced in an ABM Prism 3100-AvantSequencer. The obtained sequences were analyzed using BLAST tool to get the relative identification of eachbacterial species. PCR samples were purified, after that the bacterial extracts were subjected to thin layerchromatography for detecting the activity of extract and their componentsScreening of Bacterial metabolite with GC-MS:The active bacterial extract which was showing the maximum inhibition on cancer cell lines growth wasanalyzed by Gas Chromatography and Mass Spectrophotometer, using this technique the main compoundhaving the anticancerous activity can be detected III. RESULTS AND DISCUSSION Cancer cells were tested with Chloroform, Methanol and Ethyl acetate bacterial extracts cell viability wasevaluated by MTT assay. A gradual decrease in the viability of HCT 15 and MES-SA cancer cells were observed in for allthe bacterial extracts used in the study. The bacteria was identified as Micrococcus luteus by 16s r RNA sequencing. MTT cell growth inhibition assay was taken as in vitro measure of anticancer activity of bacterial extracts by usingdifferent cancer cell lines. Ethyl acetate extracts of bacteria are effective in inhibiting cancer cell growth of the two cancercell lines. Therefore present invitro studies on ethyl acetate bacterial extracts demonstrate the remarkable anticancerpotentials due to the presence of active principles may responsible for this anti cancer activity. Hence Micrococcus luteusEthyl acetate extracts can be used as a potent natural source of anticancer agent. The GC-MS analyses the bacterial extractand compound identified as pyrrolo (1, 2-alpha) pyrazine1,4 dione hexahydro 3-(2-methylpropyl)Isolation of pure cultures: The bacterial pure cultures were isolated by serial dilution and streak plating on nutrient agar medium and selectedbacterial colonies were sub cultured in to nutrient broth, and then the bacterial cultures were lyophilized and dissolved inchemical solvents. These bacterial extracts were dissolved in DMSO for long period further use. FIGURE 1: Isolation of pure cultureTrypan blue staining Report:The trypan blue staining was performed and the cancer cell line viability cell proliferation as shown below Table 1: Trypan blue staining % of Viability of cancer cells Cell line % viability Live cell Total cell PH count count MES-SA 81.1% 1.72×105 2.12×105 7.5 HCT 15 72% 1.728×105 2.40×105 6.9GC-MS Report: Gas chromatography and mass spectrophotometer analyses the bacterial extract and the metabolite produced bybacteria is identified as Pyrrolo [1, 2-a] pyrazine-1, 4-dione, hexahydro-3-(2-methylpropyl) www.ijmer.com 4512 | Page
  4. 4. International Journal of Modern Engineering Research (IJMER) www.ijmer.com Vol.2, Issue.6, Nov-Dec. 2012 pp-4510-4515 ISSN: 2249-6645 FIGURE 1: GC-MS analysis report with bacterial extract16s rRNA sequencing report:The selected bacterial extract sample was analyzed by using 16s rRNA sequencing, the isolate was identified as Micrococcusluteus>mt_16srRNACGCATGGTGGGTGTTGGAAAGATTTATCGGTTTTGGATGGACTCGCGGCCTATCAGCTTGTTGGTGAGGTAATGGCTCACCAAGGCGACGACGGGTAGCCGGCCTGAGAGGGTGACCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGCGCAAGCCTGATGCAGCGACGCCGCGTGAGGGATGACGGCCTTCGGGTTGTAAACCTCTTTCAGTAGGGAAGAAGCGAAAGTGACGGTACCTGCAGAAGAAGCACCGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGGTGCGAGCGTTATCCGGAATTATTGGGCGTAAAGAGCTCGTAGGCGGTTTGTCGCGTCTGTCGTGAAAGTCCGGGGCTTAACCCCGGATCTGCGGTGGGTACGGGCAGACTAGAGTGCAGTAGGGGAGACTGGAATTCCTGGTGTAGCGGTGGAATGCGCAGATATCAGGAGGAACACCGATGGCGAAGGCAGGTCTCTGGGCTGTAACTGACGCTGAGGAGCGAAAGCATGGGGAGCGAACAGGATTAGATACCCTGGTAGTCCATGCCGTAAACGTTGGGCACTAGGTGTGGGGACCATTCCACGGTTTCCGCGCCGCAGCTAACGCATTAAGTGCCCCGCCTGGGGAGTACGGCCGCAAGGCTAAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGCGGAGCATGCGGATTAATTCGATGCAACGCGAAGAACCTTACCAAGGCTTGACATGTTCTCGATCGCCGTAGAGATACGGTTTCCCCTTTGGGGCGGGTTCACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTCGTTCCATGTTGCCAGCACGTCGTGGTGGGGACTCATGGGAGACTGCCGGGGTCAACTCGGAGGAAGGTGAGGACGACGTCAAATCATCATGCCCCTTATGTCTTGGGCTTCACGCATGCTACAATGGCCGGTACAATGGGTTGCGATACTGTGAGGTGGAGCTAATCCCAAAAAGCCGGTCTCAGTTCGGATTGGGGTCTGCAACTCGACCCCMTT assay Report: From the Table 2 and graph 1: we can say that as the concentration of Chloroform, Methanol and Ethylacetate bacterial extracts increased from 20 to 120 µl/ml, the % HCT 15 cancer cell growth inhibition wasincreased from 26.19 % to 69.76 % in chloroform bacterial extract, 29.45% to 73.56 % in Methanol bacterialextract and 33.24 % to 87.34% in Ethyl acetate bacterial extract, that means they induces a cell arrest to inhibitthe growth of the HCT 15cancer cells. The Ethyl acetate bacterial extract showing the promising results www.ijmer.com 4513 | Page
  5. 5. International Journal of Modern Engineering Research (IJMER) www.ijmer.com Vol.2, Issue.6, Nov-Dec. 2012 pp-4510-4515 ISSN: 2249-6645 Table 2: Percentage of HCT 15 cell inhibition at various concentrations Concentration Percentage of HCT 15 cell inhibition µg/ml Chloroform Methanol Bacterial Ethyl acetate Bacterial extract extract Bacterial extract 20 26.19 29.45 33.24 40 32.12 38.62 41.77 60 38.47 47.56 57.33 80 45.28 58.08 64.73 100 54.11 62.9 70.65 120 69.76 73.56 87.34 Graph 1: Effect of Chloroform, Methanol and Ethyl acetate bacterial extracts on HCT 15 cancer cell growth inhibition IV. CONCLUSION Bacterial extracts of the metabolites were in vitro tested for their cytotoxic potential on various cancer cell lines.The extracts were screened on HCT 15 and MES-SA cell lines. The Ethyl acetate extract of bacterial isolate showedpromising results by MTT assay and Trypan blue staining. The isolate was identified as Micrococcus luteus sps by using 16srRNA typing, GC-MS analyses the bacterial extract and the metabolite is identified as Pyrrolo [1, 2-a] pyrazine-1,4-dione,hexahydro-3-(2-methylpropyl) showing promising results were obtained with anti cancerous activity V. ACKNOWLEDGEMENTS SAI LAKSHMAN MITHUN.V would like to thank Department of Biotechnology, DVR& Dr.HSMIC College of Technology, Kanchikacherla, and Andhra Pradesh for their cooperation to complete this workFor Masters degree REFERENCES[1] Angel TreasaThomas , Josyula Venkata Rao, Volety Mallikarjuna Subramanian , Hariharapura Raghu Chandrasekhar, Naseer Maliyakkal1, Tukaram Kedar Kisan, Alex Joseph, Nayanabhirama Udupa, In vitro anticancer activity of microbial isolates from diverse habitats, Brazilian Journal of Pharmaceutical Sciences, vol. 47, n. 2, apr./jun., 2011[2] B Dhorajiya, M Malani, B Dholakiya, Extraction and Preservation Protocol of Anti-Cancer Agents from Marine World, Chemical Sciences Journal, CSJ-38, accepted version, Oct 22, 2011[3] Angel.T.T, Venkata rao.J, Jesilm.A, Subrahmanyam.V.M, Venkatesh.K.B, Udupa.N. Antimicrobial profile of extremophiles from aqua to terrestrial habitats. Pharmacology online, v.1, p.111-126, 2009.[4] T. Mossman, J. Immunol. Methods; 1983, 65, 55. www.ijmer.com 4514 | Page
  6. 6. International Journal of Modern Engineering Research (IJMER) www.ijmer.com Vol.2, Issue.6, Nov-Dec. 2012 pp-4510-4515 ISSN: 2249-6645[5] Sanjay patel. Nirav gheewala, Ashok suthar, Anand shah, In-vitro cytotoxicity activity of solanum nigrum extract against Hela cell line and vero cell line, International Journal of Pharmacy and Pharmaceutical Sciences, Vol. 1, Suppl 1, Nov.-Dec. 2009[6] CLARDY. J. Discovery of new compounds in nature. Proc. Amer. Phil. Soc., v.151, p.201-210, 2007.[7] BUTLER, M.S. Natural products to drugs: natural products derived compounds in clinical trials. Nat. Prod. Rep., v.25, p.475-516, 2008.[8] HARLEY, J.P.; PRESCOTT, L.M. Laboratory exercises in microbiology. 5. ed. Columbus: The MC Graw Hill Companies, 2002. P.13-16, 125-207.[9] ATTA–UR-RAHMAN.; IQBAL, C.E.; WILLIAM, J.T. Bioassay techniques for drug development. Amsterdam: Harwood Academic Publishers, 2001. P.1-5[10] Phillips HJ and Terryberry JE. Counting actively metabolizing tissue cultured cells. Cell Research 1957; 13: 341-347.[11] Masters RW. Animal cell culture, Trypan Blue Assay sop. 3rd ed. 2000, p. 1 – 3[12] Skehan P, Storeng R, Scudiero D, Monks A, McMahon J, Vistica D et al. Evaluation of Colorimetric Protein and Biomass Stains for Assaying Drug Effects upon Human Tumor Cell Lines. Proceedings of the American Association for Cancer Research 1989; 30: 612.[13] Skehan P, Storeng R, Scudiero D, Monks A, McMahon J, Vistica D et al. New Colorimetric Cytotoxicity Assay for Anticancer-Drug Screening. Journal National Cancer Institute 1990; 82(13), 1107-1112.[14] Masters RW. Animal cell culture, Cytotoxicity and viability assays. 3rd ed. 2000, p. 202-203.[15] JEFFREY, M.E.; LINDA, S.A.; ANDREW, O.M. A rapid and simple MTT based spectrophotometric assay for determining drug sensitivity in monolayer culture. Tissue Cult. Meth, v.11, p.15-17.[16] McCloud TG, 2010. High Throughput Extraction of Plant, Marine and Fungal Specimens for preservation of Biologically Active Molecules. Marine Drugs, 15: 4526-4563[17] MARIANNA, J.; JAMES, P.K.; RONALD, R.R. Macquarimicins, microbial metabolites from Micromonospora I. Discovery, taxonomy, fermentation and biological properties. J. Antiriot., v.48, p.462-466, 1995[18] Newman DJ, Cragg GM, 2007. Natural products as sources of new drugs over the last 25 years. Natural Products, 70: 461–477.[19] Rajeev Kumar Jha, and Xu Zi-rong, 2004 Biomedical Compounds from Marine organisms, Marine drugs 2,123-124[20] Alejandro M.S. MAYER and Kirk R. GUSTAFSON, Marine pharmacology in 2000: Anti tumor and cytotoxic compounds, International journal of Cancer: 105, 291–299 (2003) www.ijmer.com 4515 | Page

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